Uncontrolled inflammation is at the root of many diseases, including those affecting the cardiovascular system such as atherosclerosis, sepsis, and autoimmune disorders. Recent advances in molecular understanding of these diseases indicate that MyD88 is a key intracellular adaptor that integrates multiple intracellular signaling cascades activated by proinflammatory cues. These stimuli are sensed by Toll-like receptors (TLRs), the mainstays of innate immunity and inflammation. Ultimately, they induce translocation of stress-responsive transcription factors to the nucleus and subsequent activation of hundreds of genes that encode mediators of inflammation. I hypothesize that targeting MyD88 function would block the majority of TLR-initiated signaling pathways, thereby suppressing proinflammatory gene expression. The first aim of this proposal focuses on the design, production, and testing of a novel cell-penetrating (CP) protein, CP-MyD88 TIR, to analyze its effect in murine macrophages challenged with the TLR4 ligand lipopolysaccharide (LPS). The mechanism by which CP-MyD88 TIR inhibits TLR4 signaling will then be characterized. A series of CP-MyD88 TIR mutants will be used to establish the role of the previously mapped sites in interacting with TIR domain of MyD88-like adaptor (Mai) as compared to that in IL1 beta RAcP TIR domain. In the second aim, the functionally characterized CP-MyD88 TIR protein will be tested in an animal model of endotoxic shock. The results of these studies will yield new mechanistic insights into the interaction of MyD88 with proinflammatory signal transducers. It will also represent a novel platform for preclinical testing of innovative therapy to counteract uncontrolled inflammation induced by LPS and other microbial agents that cause cardiovascular collapse in sepsis. Relevance: Uncontrolled inflammation causes organ injury in many diseases, including sepsis, atherosclerosis, and autoimmune disorders. We will develop a new type of protein reagent that will enter cells and block the chain of reactions by which inflammation is "turned on." In this way, it will suppress uncontrolled inflammation and limit the damage it causes.